Platelets Inhibit Methicillin-Resistant Staphylococcus aureus by Inducing Hydroxyl Radical-Mediated Apoptosis-Like Cell Death

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant bacteria and poses a significant threat to human health. Due to the emergence of multidrug resistance, limited drugs are available for the treatment of MRSA infections. In recent years, platelets have been reported to play important roles in inflammation and immune responses, in addition to their functions in blood hemostasis and clotting. We and other researchers have previously reported that platelets can inhibit Staphylococcus aureus growth. However, it remained unclear whether platelets have the same antibacterial effect on drug-resistant strains. In this study, we hypothesized that platelets may also inhibit the growth of MRSA; the results confirmed that platelets significantly inhibited the growth of MRSA in vitro. In a murine model of MRSA infection, we found that a platelet transfusion alleviated the symptoms of MRSA infection; in contrast, depletion of platelets aggravated infective symptoms. Moreover, we observed an overproduction of hydroxyl radicals in MRSA following platelet treatment, which induced apoptosis-like death of MRSA. Our findings demonstrate that platelets can inhibit MRSA growth by promoting the overproduction of hydroxyl radicals and inducing apoptosis-like death. IMPORTANCE The widespread use of antibiotics has led to the emergence of drug-resistant bacteria, particularly multidrug-resistant bacteria. MRSA is the most common drug-resistant bacterium that causes suppurative infections in humans. As only a limited number of drugs are available to treat the infections caused by drug-resistant pathogens, it is imperative to develop novel and effective biological agents for treating MRSA infections. This is the first study to show that platelets can inhibit MRSA growth in vitro and in vivo. Our results revealed that platelets enhanced the production of hydroxyl radicals in MRSA, which induced a series of apoptosis hallmarks in MRSA, including DNA fragmentation, chromosome condensation, phosphatidylserine exposure, membrane potential depolarization, and increased intracellular caspase activity. These findings may further our understanding of platelet function.

against S. aureus. The manuscript is well written and the data presented support the conclusions. Some sections of the methods are either lacking or unclear, for example the phenotypical determination of methicillin susceptibility and the mice infection and blood collection protocols.
Specific comments: Introduction, lines 60-62: there is some confusion in this sentence as the human immunodeficiency virus is included among major pathogenic bacteria Introduction, lines 62-63: the percentage of MRSA among clinical S. aureus isolates varies dramatically in different regions of the world. The 20-50% range reported should be intended as an approximation for most, but not all, countries. Results, lines 98-105, 111-115, and Figure 1: It is not clear how methicillin resistance was confirmed in the study strains, and how CFU counts were estimated over the course of the survival curves. Phenotypic resistance (MIC of cefoxitin in Mueller-Hinton medium or cefoxitin disc diffusion diameter) should be reported for each strain used. The antibiotic concentration should also be indicated in mg/L. Additionally, the killing/survival cures should show several time points during at least a 24h time span, rather than just one time point (figure 1 A, B). Similarly, time 0 values should be shown for panels F and H. Finally, log10 values in the 12 to 13 range for the CFU/ml seem too high (panel H), and do not seem to match with the OD600 values (panel F). Similar considerations apply to figure S1. Results, lines 133-136, and figure 2: It would be useful to know if steps were employed to ensure possible aggregates of MRSA were separated before counting (washing, addition of antiaggregating agent, etc.). OD depends more on the number of the number of floating particles, than on their size. Methods, lines 378-383: it is not clear how many times and how much blood was collected from mice: was each mouse sampled four times in the span of 24h, or were different mice sampled at different time points? What volume of blood was collected each time? Was local anesthesia performed? This should be described in better detail, including also adherence with the author's Institution laboratory animals guidelines.

Reviewer #2 (Comments for the Author):
See attached file Staff Comments:

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If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website. What are these strains? Are there significant genomic differences between these strains and USA300 or Newman? 5. 'Dead' MRSA classified in Figure S1F-g don't make sense. How do the authors know the bacteria are dead? Optical opacity? 6. When the authors describe a severe decrease in OD for strains incubated with platelets, how do they differentiate bacterial death from aggregation? (Similar to the decrease in OD found during agglutination). 7. Is there fibrinogen and/or fibrin in these extracts/platelet supernatants? If so this should be taken into account when measuring phenotypes. SA is known to aggregate with platelets, via ClfA and FnbpA/B 8. There are some important controls missing. For example, in Figure 6, B-F there need to be positive controls for membrane disruption and caspase activity, without which these data don't make sense. 9. The animal model with platelet transfusion is a little confusing. If 10 9 platelets were injected into a host it is likely that there will be infiltration of some immune cells! Also, there's not much of an increase in platelets either? 10. The studies with anti-CD42b treated mice cannot be taken into consideration without the appropriate controls. It would seem that the previously used model of platelet transfusion would be a perfect control to show the complementation of phenotypes. 11. Is it just OH? How are the other ROS species ruled out? 12. Why are the axes in the FloJo images in Figure 6 removed? And once again, there needs to be a positive control for PS exposure. How about E. coli! (Since they mention it). Minor Comments  There are numerous typos and grammatical errors that need to be fixed.  It is a far reach to show these results and claim that the manuscript provides evidence for novel antibacterial agents or treatments against drug resistant bacteria. Authors should refrain from making these statements. They are unnecessary and not supported by the data presented.  It should be specified whether the t-tests are paired or unpaired and whether all results were analyzed with t-tests. Also if t-tests are used then specific P values should be provided.

List of Responses
The reviewers' comments concerning our manuscript, titled "Platelets Inhibit

Methicillin-resistant Staphylococcus aureus by Inducing Hydroxyl Radical-
mediated Apoptosis-like Cell Death (Spectrum 02441-21)," have helped us in improving the quality of this manuscript. We have gone through all the comments carefully and tried our best to incorporate the changes recommended. All the revised parts are marked in yellow in the revised manuscript. Point-to-point responses to the reviewer's comments are given below.

Introduction, lines 60-62: there is some confusion in this sentence as the human immunodeficiency virus is included among major pathogenic bacteria.
Response: We have amended this sentence as: MRSA ranks first among the three major pathogenic microorganisms prevalent worldwide (Mycobacterium tuberculosis, human immunodeficiency virus, and MRSA) and accounts for 20-50% of clinically isolated S. aureus in many regions of the world. The revised sentence has been added in page 4, lines 59-62.

Introduction, lines 62-63: the percentage of MRSA among clinical S. aureus isolates varies dramatically in different regions of the world. The 20-50% range reported should be intended as an approximation for most, but not all, countries.
Response: We have revised this sentence as "MRSA ranks first among the three major

Additionally, the killing/survival cures should show several time points during at least a 24h time span, rather than just one time point (figure 1 A, B). Similarly, time 0 values should be shown for panels F and H.
Response: We observed that the turbidity of the PLT-MRSA group (indicating MRSA growth) was much lower than that of the MRSA group. The optical density at 600 nm (OD600) was detected after co-culture for 10 h, and we found that OD600 decreased significantly, indicating that the platelets had achieved an excellent bacteriostatic effect at 10 h. These results are depicted in Fig. 1A and B. Then, we established the survival curves of platelet-inhibiting MRSA by measuring the OD600 value and colony counts using plates (CFU/mL) every 2 h for a 24 h period. The survival curves are shown in Fig. 1E and G. Additionally, the OD600 values at 0 h has been added to Fig.   1F and H.

Finally, log10 values in the 12 to 13 range for the CFU/ml seem too high (panel H), and do not seem to match with the OD600 values (panel F). Similar
considerations apply to figure S1.
Response: To confirm bacterial colony count results, we performed colony counts of the bacterial suspension at each time point again; results showed that the maximum colony counts (CFU/ml) of log10 were between 10 and 11 (Fig. 1H, S2B and S2C). Response: In order to ensure the accuracy of the OD600 value and colony count results, washing was carried out prior to detection to prevent MRSA aggregation. This information has been added to page 19, lines 389-391 of the Methods section. Major Comments:

The reasoning behind why the authors decided to do these studies doesn't make sense. They say that they did studies with S. aureus and showed killing mediated by platelets, so they wanted to know if MRSA does it too. Is there reason to believe the sccmec element would alter this phenotype? Why would it be any different?
Response: In our previous studies, we found that platelets directly regulate DNA

Neutrophils, macrophages and other immune cells also produce hydroxyl radicals, but are not able to eliminate S. aureus. (Or MRSA) So why are only platelets so efficient at doing this? Is the concentration of OH released by platelets higher? If so this should be discussed.
Response: In this study, we demonstrated that the platelets co-cultured with MRSA led to excessive generation of OH • in MRSA itself, which led to apoptosis-like death of MRSA. We therefore primarily detected the OH • produced by MRSA, rather than that produced from the platelet secretion. The exclusion of the possible interference of OH • from the platelet secretion was mainly based on the following evidence: a. The results shown in Fig. S4A, S4B, and Fig. 4A provide evidence that MRSA suffered severe oxidative stress after platelet treatment. b. In order to inhibit the ability of platelets to produce OH • , platelet lysates were prepared in advance and then applied to MRSA. Results showed that the increased levels of OH • in MRSA were consistent with those in the platelets-treated group (Fig. S5F and G). c. In the revised manuscript, mitomycin C (MMC) has been mentioned as the positive control. Results showed that the intracellular OH • levels were also significantly increased after the treatment of MMC (5μg/mL) with MRSA for 4 h (Fig. 4B and C).

The authors would benefit from showing the OH phenotype when S. aureus is incubated with OH radicals from a chemical source (reagent).
Response: We conducted experiments by including H2O2 (10 mM) into the culture medium of MRSA, and the results showed that H2O2 significantly inhibited the growth of MRSA (Fig. S2E and F).

Why isn't USA300, the most well-known MRSA strain used here as a reference?
What are these strains? Are there significant genomic differences between these strains and USA300 or Newman?

'Dead' MRSA classified in Figure S1F-g don't make sense. How do the authors know the bacteria are dead? Optical opacity?
Response: In this revised manuscript, we added the bacterial colony count after platelet treatment; result showed that the bacterial colony count of MRSA in the platelet-treated group was significantly reduced (Fig. S2C). In addition, detection of trypan blue staining using an oil microscope showed that several dead MRSA were surrounded by lysed platelets (Fig. S2D). These results indicate that platelet treatment caused MRSA death.

When the authors describe a severe decrease in OD for strains incubated with platelets, how do they differentiate bacterial death from aggregation? (Similar to the decrease in OD found during agglutination).
Response: In order to ensure the accuracy of the OD600 value, washing was carried out before detection to prevent MRSA aggregation. We performed bacterial colony counting by plate to demonstrate the antibacterial effect of platelets ( Fig. 1H and S2C).
This experimental detail has been added to page 19, lines 389-391 of the Method section.

Is there fibrinogen and/or fibrin in these extracts/platelet supernatants? If so this should be taken into account when measuring phenotypes. SA is known to aggregate with platelets, via ClfA and FnbpA/B.
Response: Although human apheresis platelets used in the experiment were adequately washed to remove the plasma component, a little fibrinogen may still be have remained in the co-culture system due to its expression in the platelets. It has been reported that platelet adhesion and encapsulation of S. aureus is an important step in platelet sterilization (4). Platelet-derived antimicrobial peptides play a key antibacterial role after adhering to and wrapping bacteria (5,6). Likewise, in our study, trypan blue staining results, detected using an oil microscope, showed that several dead MRSA were surrounded by lysed platelets (Fig. S2D). Taken together, the bacterial colony count and trypan blue staining results illustrated the antibacterial effect of platelets on MRSA (Fig. 1H, S2C and S2D). This information has been added to the discussion part in page 16-17, lines 325-335. Figure 6, B-F there needs to be positive controls for membrane disruption and caspase activity, without which these data don't make sense.

There are some important controls missing. For example, in
Response: Mitomycin C (MMC) has been reported to induce apoptotic-like hallmarks in model eukaryotic systems and E. coli (7). This detailed description has been added to page 11, lines 214-216. Hence, MMC was used as the positive control. The related indicators of apoptosis, including DNA fragmentation ( Fig. 5B and C), phosphatidylserine exposure ( Fig. 6A and B), membrane potential depolarization ( Fig.   6C and D), and increased intracellular caspase activity of bacteria ( Fig. 6E and F), were detected after MRSA was treated with MMC (5μg/mL) for 4 h.

The animal model with platelet transfusion is a little confusing. If 10 9 platelets were injected into a host it is likely that there will be infiltration of some immune cells! Also, there's not much of an increase in platelets either?
Response: a. In our study, blood collected from C57BL/6 mice was centrifuged twice (150 × g each time) for 10 min, and red and white blood cells were removed before platelet transfusion. A third centrifugation step (750 × g, 15 min) was performed to obtain platelet precipitates, which were then suspended in PBS. The detailed preparation of mouse platelets is described in page 20, lines 405-411. After three centrifugation steps, most white and red blood cells were removed, and platelet suspension cell count results showed that the white blood cell count was not detected (Fig. S3G). These findings indicate that the effect of very few immune cells on platelet transfusion was very slight.
b. Platelet infusion in mice was performed after 6 h of MRSA infection, and the earliest time point for blood collection from mice was 8 h after infection. Blood was collected from the mice 2 h after platelet infusion ( Fig. 2A to E). It has been reported that platelets serve as "sentinels" for tissue damage and microbial invasion, monitor the integrity of blood vessels, and help the body build an effective immune system (8).
During infection, platelets quickly travel to the site of infection in large numbers (9).
This detailed description has been added to page 4, lines 68-70. Thus, in this experiment, as the mice were in a state of acute infection, transfused platelets may quickly reach the infected site after platelet transfusion. As a result, the number of platelets in the peripheral blood did not increase significantly two hours later (Fig.   2C), but the infection status of the mice was alleviated (Fig. 2F to H).

The studies with anti-CD42b treated mice cannot be taken into consideration without the appropriate controls. It would seem that the previously used model of platelet transfusion would be a perfect control to show the complementation of phenotypes.
Response: To verify that platelets play a certain antimicrobial role in vivo, we conducted two experiments-platelet transfusion to elevate platelet levels and anti-CD42b antibody treatment to deplete platelets. This two-experiment design was used as a parallel control to support the antibacterial effect of platelets in mice. The results are shown in Figures 2 and 3.

Is it just OH? How are the other ROS species ruled out?
Response: In general, ROS consist of three main types: superoxide (O2 -), hydrogen (H2O2), and hydroxyl radicals (OH • ) (10). As shown in figures 3 and 4 below, when cells are subjected to oxidative stress, O2produced by the oxidative respiration chain is converted into H2O2 under the action of superoxide dismutase. H2O2 in the presence of Fe 2+ generates OH • through the Fenton reaction (10)(11)(12). So, both O2and H2O2 can eventually be converted into OH • in vivo. OH • is considered the most toxic and deadly of the three types. As shown in Figure 5 below, the highly destructive OH • acts as an "executor" of cell death, which directly damages DNA, lipids, and proteins, and ultimately leads to cell death (13). Therefore, we took the most important OH • into account. The new statement has been added to page 9-10, lines 174-183.    Figure 6   Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.

Why are the axes in the FloJo images in
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